This invention relates to the field of clinical assay techniques and involves the measurement of low density lipoprotein cholesterol.
Lipoproteins are complex particles comprising proteins and lipids which are found in the circulatory system. One of their functions is to carry water insoluble substances, such as cholesterol and cholesterol esters, for eventual cellular utilization. While all cells require cholesterol for growth, the excess accumulation of cholesterol by cells can lead to certain diseases including atherosclerosis.
There are a variety of classes of lipoproteins in serum which can be classified by their density. These classes include very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). All of these lipoproteins contain varying amounts of cholesterol. A total serum cholesterol determination is a complex sum of the amount that each lipoprotein contributes to the total lipoprotein population of the serum.
While it is known that the amount of total serum cholesterol can be correlated with the incidence of atherosclerosis, evidence from studies of recent years has shown that specific lipoprotein types are more closely associated with the progression of heart disease, including atherosclerosis, than others. More recent studies have implicated LDL as the class of lipoproteins responsible for the accumulation of cholesterol in the cells whereas HDL has been shown to be active in the removal of excess cholesterol from cells. Accordingly, various systems have been proposed for the measurement of cholesterol bearing lipoproteins in general and LDL in particular.
Amorphous silica, i.e. that form of SiO.sub.2 which lacks a crystal structure, has been used as an adsorbant since at least as early as World War I when it was considered for use as an absorbant in gas masks. Amorphous silica is broadly divided into three categories: vitreous silica or glass made by fusing quartz; silica M made by irradiating either amorphous or crystalline silica with high speed neutrons and microporous silica. The microparticulate silicas include pyrogenic silicas and silicas precipitated from aqueous solution. Pyrogenic silicas are formed at high temperature by condensation of SiO.sub.2 from the vapor phase, or at lower temperature by chemical reaction in the vapor phase followed by condensation.
Silica formed in aqueous solution can occur as sols, gels or particles. A gel has a three-dimensional, continuous structure, whereas a sol is a stable dispersion of fine particles.
Silica gels are classified into three types. Regular density gel is made by gelling in an acid medium, which gives very small particles with high surface area (750-800 m.sup.2 /g). The average pore diameter is 22-26 .ANG., and the pore volume is 0.37-0.40 mL/g. Regular density gel contains about 6 wt % water as surface hydroxyl groups, which imparts a high propensity for adsorption of water and other polar molecules. Regular density gel exhibits a high selectivity for polar molecules and possesses a large percentage of small pores. Intermediate density silica has a lower surface area (300-350 mg.sup.2 /g) but larger pore volume (0.5-1.1 mL/g). The average pore diameter is 120-180 .ANG. in diameter and the particles are larger than those of regular density gel. Because of the large pore size, intermediate density gel has a large capacity for water absorption at high humidities. Low density silica gel has a lower surface area (&lt;200 m.sup.2 /g), larger pore diameter (&gt;180 .ANG.) and a larger pore volume (&gt;1.5 mL/g) than the other types. It is usually prepared as a very fine powder of extremely low density. When silica is used as an adsorbant, the pore structure determines the gel adsorption capacity. Pores are characterized by specific surface area, specific pore volume (total volume of pores per gram of solid), average pore diameter, pore size distribution and the degree to which entrance to larger pores is restricted by small pores. These parameters are derived from gas or vapor adsorption isotherms, mercury penetration studies, low angle X-ray scattering, electron microscopy, and gas permeability or measurement of the volume of imbibed liquid.
The most common way of preparing silica gel involves acidification of sodium silicate to a pH less than about 10. Silica can be gelled in spherical form by spray-drying, or by spraying droplets onto an immiscible liquid.
Precipitated silica (also called particulate silica) is composed of aggregates of ultimate particles of colloidal size that have not become linked in a massive gel network. Precipitated silicas are either formed from the vapor phase (fumed or pyrogenic silicas) or by precipitation from solution. In the preparation of pyrogenic or fumed silica, sand is vaporized at about 2000.degree. C. On cooling, anhydrous amorphous silica powders form in the presence of a reducing agent such as coke. The amorphous silica sublimes at about 1500.degree. C. to provide Si which is then oxidized to produce particulate SiO.sub.2. Pyrogenic or fumed silica is typically used as a thixotropic agent in polyester-glass reinforced plastics; as a reducing and thickening agent in rubber, plastics, silicone and epoxy resins as well as a thickening and gelling agent.
Pure silica is composed of the elements silicon and oxygen. Materials are still referred to as "silicas" after metals, metal oxides or metal salts are added; e.g. flint is a silica with added iron oxide. Glass has a defined composition between (K,Na).sub.2 O, (Ca,Pb), 6SiO.sub.2 and 5(K,Na).sub.2 O, 7(Ca,Pb)O and 36SiO.sub.2 with a general formula of (K,Na)O--Si.sub.n O.sub.2n-1 (CaPb)O--Si.sub.n O.sub.2n-1 --O(K,Na). While all silica based glasses can be called silicas, not all silicas are glass. The HDL adsorbant materials useful in the present invention are porous silica or silicates as these terms are used in their broadest sense.
Microporous silica gels are obtained by heating a hydrated gel at 1000.degree. C. for about 10 hours. Siliceous materials can be made with extremely small pores such as is the case with impervious silica, porous glass and silica used as an adsorbant for certain specific materials. The ability of a material to be adsorbed is determined by the surface composition and pore size of the silica gel. The present invention is concerned with the use of large pore silicas and silicates such as microporous silica, silica gel and controlled pore glass as selective adsorbant materials for HDL from blood serum or plasma.
U.S. Pat. No. 5,141,872 discloses the use of fumed silica for the adsorption of lipoproteins from plasma. The patentees point out that this procedure was known before their invention but claim the improvement of selectively desorbing HDL from the fumed silica by incubating with a detergent containing formulation. Commercially available fumed silicas such as CAB-O-SIL from Cabot Company and Aerosol from Degussa are mentioned as being useful in this procedure.
The diameters of the LDL and VLDL particles are estimated at 220-250 .ANG. and 300-800 .ANG. respectively with chylomicrons being somewhat larger. Since the dimensions of a fumed silica such as Aerosol 380 are less than about 70 .ANG. and the HDL particle is estimated at 100 to 150 .ANG. in diameter, it can be concluded that this binding of lipoproteins as disclosed in U.S. Pat. No. 5,141,872 is based solely on non-specific surface adsorption. Particle size exclusion of the relatively larger lipoprotein particles is not a factor in that method since LDL and VLDL particles are too large to fit in any pores which may exist in the 70 .ANG. particles. This prior art technique achieves its selectivity by desorption in a separate step, whereas the present invention involves the selective adsorption of the smaller HDL particles by the silica gel which, when combined with a mechanism for the separation of VLDL and chylomicrons, provides a fluid sample which can be analyzed for the remaining LDL without further treatment.